The scientific scope of this revision application is relevant to and goes beyond the aims of NIH AR46568-09 Physiologic Loading for Cartilage Tissue Engineering (9/1/09- 8/31/13, PI: Hung). This BIRT proposal aims to incorporate proteomics as an important tool for optimizing culture protocols for tissue engineering of cartilage with mechanical function similar to the native tissue. The parent grant utilizes a cell source comprised of cartilage cells called chondrocytes, which are expanded in two-dimensional (2D) monolayer tissue culture with a growth factor cocktail before seeding in three- dimensional (3D) hydrogel scaffolds that are implanted into cartilage defects in a canine knee in vivo model. Toward this objective, a new multidisciplinary collaboration will be established between two researchers at Columbia University, the Principal Investigator, Dr. Clark T. Hung, Professor of Biomedical Engineering, with expertise in cartilage mechanobiology and tissue engineering, and Co-Investigator, Dr. Lewis Brown, Director of the Comparative Proteomics Center, Department of Biological Sciences with expertise in proteomics. Additionally, Dr. Mikko Lammi, Professor of Biochemistry, Department of Biosciences, Kuopio University Finland with expertise in chondrocyte biology including proteomic analyses of chondrocytes will serve as a Consultant. The investigators have no formal collaborations or grants together. The BIRT application will pursue the following specific aims: Specific Aim 1: To use proteomic analyses to screen for biomarkers in chondrocytes expanded in 2D with a defined chemical growth factor cocktail 1 osmotic loading that can be used to identify those cells that have the greatest potential to produce articular cartilage when seeded and cultured in 3D engineered constructs as measured by functional measures prioritized in descending order of material, biochemical and histological properties (see Figure 1). Specific Aim 2: Repeat Aim 1 on synovium-derived mesenchymal stem cells that have recently been shown in the literature and in our preliminary data to be a relevant stem cell source for cartilage repair. An ability to identify biomarkers (using comparative proteomics) that can act as predictors of cell cultures with a high capacity to form functional engineered cartilage tissues will permit optimization of protocols for cartilage tissue engineering efforts using a variety of cell sources. PUBLIC HEALTH RELEVANCE: Arthritis costs $128 Billion annually in the United States. The inability of articular cartilage to heal has led to significant efforts to develop cell-based therapies and tissue engineering strategies aimed at cartilage repair. Culturing of cells on a two dimensional (2D) tissue culture dish provides a platform to increase cell number (expansion) as well as an opportunity to prime cells with chemical and physical stimuli that can induce cell differentiation toward a desired lineage (e.g., chondrogenic potential). An ability to identify biomarkers that can act as predictors of cells with a high capacity to form functional engineered cartilage tissues will permit optimization of protocols for cartilage tissue engineering efforts using a variety of cell sources.